Plastid Evolution - Sektion Biologie

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Plastid EvolutionSven B. Gould, Ross F. Waller,and Geoffrey I. McFaddenSchool of Botany, University of Melbourne, Parkville VIC-3010, Australia;email: [email protected], [email protected], [email protected]

Annu. Rev. Plant Biol. 2008. 59:491–517

The Annual Review of Plant Biology is online atplant.annualreviews.org

This article’s doi:10.1146/annurev.arplant.59.032607.092915

Copyright c© 2008 by Annual Reviews.All rights reserved

1543-5008/08/0602-0491$20.00

Key Words

secondary/tertiary endosymbiosis, complex plastids, proteintargeting, genome evolution, intracellular gene transfer, plastidbiochemistry

AbstractThe ancestors of modern cyanobacteria invented O2-generatingphotosynthesis some 3.6 billion years ago. The conversion of wa-ter and CO2 into energy-rich sugars and O2 slowly transformedthe planet, eventually creating the biosphere as we know it today.Eukaryotes didn’t invent photosynthesis; they co-opted it fromprokaryotes by engulfing and stably integrating a photoautotrophicprokaryote in a process known as primary endosymbiosis. After ap-proximately a billion of years of coevolution, the eukaryotic hostand its endosymbiont have achieved an extraordinary level of inte-gration and have spawned a bewildering array of primary producersthat now underpin life on land and in the water. No partnership hasbeen more important to life on earth. Secondary endosymbioses havecreated additional autotrophic eukaryotic lineages that include keyorganisms in the marine environment. Some of these organisms havesubsequently reverted to heterotrophic lifestyles, becoming signifi-cant pathogens, microscopic predators, and consumers. We reviewthe origins, integration, and functions of the different plastid typeswith special emphasis on their biochemical abilities, transfer of genesto the host, and the back supply of proteins to the endosymbiont.

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Contents

INTRODUCTION. . . . . . . . . . . . . . . . . 492FROM FREEDOM TO SLAVERY:

OUTLININGENDOSYMBIOTIC STEPS . . . . . 492Primary Endosymbiosis . . . . . . . . . . . 493Eukaryotic Endosymbiosis . . . . . . . . 495Nature’s Playground:

The Evolution Continues . . . . . . 498PREPROTEIN TARGETING . . . . . . 501

Targeting to Primary Plastids . . . . . 501Targeting Into and Within

Secondary Plastids . . . . . . . . . . . . . 503BIOCHEMICAL PATHWAYS . . . . . . 505

Starch Synthesis . . . . . . . . . . . . . . . . . . 505Isopentenyl Diphosphate

(Isoprenoid Precursor)Synthesis . . . . . . . . . . . . . . . . . . . . . . 506

Heme Synthesis . . . . . . . . . . . . . . . . . . 507Aromatic Amino Acid Synthesis . . . 508Fe-S Clusters . . . . . . . . . . . . . . . . . . . . 508

INTRODUCTION

In nature the counterpart of chaos is not cos-mos, but evolution. The spark of life was ini-tially a chemical one, leading to the synthesisof the first molecules. Some of these persistedand evolved in a precellular period, perhapssimilar to that described in the model of theRNA world, leading to the first prokaryoticlife approximately 3.5 to 4 billion years ago(48, 74). The invention of oxygenic photosyn-thesis by prokaryotic cyanobacteria approxi-mately 500 million years later was the nextmajor achievement of biological evolution. Ithad a major impact on the earth by enrichingthe atmosphere with O2 to a level that trans-formed the geochemistry of the planet.

The first molecular carbon skeletons typ-ical of cyanobacteria can be identified instrata from approximately 2.75 billion yearsago (15). At the same time a novel mineralknown as hematite (Fe2O3), which can formonly in the presence of a minimum criticalconcentration of oxygen, began to appear.

These geological indices testify to an ever-increasing concentration of atmospheric oxy-gen due to photosynthetic activity. Photosyn-thesis was also the evolutionary trigger for thesweeping diversification of O2-dependent life.Indeed, oxygen has become critical for mostliving things, acting as an acceptor for theelectrons released from carbon-carbon bondsthat were ultimately created using energy cap-tured by photosynthesis. Thus, a byproduct ofphotosynthesis (oxygen) became an essentialcomponent for the burning of the sugars pro-duced by photosynthesis. The balance of thebiosphere was born.

Nineteenth century microscopists (Sachs,Altmann, and Schimper) recognized the semi-autonomous nature and bacterial-like stainingproperties of chloroplasts (then known aschlorophyll bodies) and mitochondria (thenknown as cell granules) (4, 106), but it tookanother 15 years before Mereschkowsky syn-thesized these observations into the theorythat chloroplasts are derived from cyanobac-teria (81, 109). Margulis later formalized theTheory of Endosymbiosis, which posits thatplastids and mitochondria of eukaryotic cellsderive from bacterial endosymbionts (71).

FROM FREEDOM TO SLAVERY:OUTLINING ENDOSYMBIOTICSTEPS

As far as we know, all eukaryotes have mi-tochondria (or modified, anaerobic forms ofmitochondria known as hydrogenosomes ormitosomes), and the establishment of thispartnership is generally regarded as inte-gral to the origin of eukaryotes (123). Theacquisition of plastids by eukaryotes oc-curred later, after the establishment of a di-versity of heterotrophic eukaryotic lineages,one of which adopted a cyanobacterium-like endosymbiont to acquire photosynthe-sis and become autotrophic. We refer to aninitial plastid-creating endosymbiosis as theprimary endosymbiosis. Secondary (or eu-karyotic) endosymbiosis refers to subsequentendosymbiotic events in which the progeny

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of the primary endosymbiotic partnershipbecome endosymbionts within other het-erotrophic eukaryotes, thus transferring thecaptured cyanobacterial symbiont laterallyamong eukaryotes. Subsequently, the progenyof these secondary endosymbiotic partner-ships have become endosymbionts in othereukaryotes, creating tertiary endosymbioses,to weave an extraordinarily complex set ofendosymbiotic relationships of cells withincells within cells within cells (Figure 1). Inthis review we examine the cell biology ofthese endosymbiotic events and examine howthe various compartments and genomes ofthese extraordinary chimeras cooperate as asingle cell, albeit one made up of parts frommultiple individual cells.

Primary Endosymbiosis

The endosymbiotic integration of a free-living, cyanobacterial-like prokaryote into aeukaryotic host produced three major au-totrophic lineages: the glaucophytes, thegreen algae (and their descendants, theplants), and the red algae (2, 46) (Figure 1).Plastids in these primary endosymbionts arecharacterized by having two bounding mem-branes, which are derived from the two mem-branes (plasma membrane and outer mem-brane) of the Gram-negative cyanobacterium(17, 20). If a phagocytotic membrane sur-rounded the symbiont when it was first inter-nalized by the host, it has disappeared with-out a trace (20). The main lines of evidencesupporting homology between the outer en-velope membrane and the outer membraneof a cyanobacterium are (a) the presence ofgalactolipids (52), (b) the presence of β-barrelproteins in both membranes (110), and (c) theoccurrence of peptidoglycan (or rudiments ofpeptidoglycan synthesis machinery) beneaththese membranes (117).

Phylogenetic analyses suggest that theglaucophytes were the first primary endosym-biotic lineage to diverge, some 550 mya,and that the red and green algae divergedlater (75, 82, 103). Plants, which probably

diverged from their green algal ancestorsapproximately 400 to 475 mya (36), subse-quently conquered the terrestrial environ-ment, paving the way for animals to followthem onto land. In accordance with this se-quence, plastids in the glaucophytes (whichare sometimes referred to as cyanelles but aredefinitely plastids) most resemble theircyanobacterial ancestors in that they re-tain a peptidoglycan, wall-like layer betweenthe inner and outer envelope membranes(57). Additionally, the thylakoids inside theglaucophyte plastid stroma are studded withphycobilisomes that are identical to thoseof cyanobacteria, and the composition ofthe oxygen-evolving enhancer complex isalso very similar to that of free-livingcyanobacteria (117). Rhodophyte plastids alsouse phycobilins in protein-based light har-vesting antenna (phycobilisomes), but theirplastids have apparently lost the peptido-glycan wall (31). The green algal/plantlineage plastids are the most derived in the pri-mary endosymbiosis lineage. Phycobilisomeswere replaced by chlorophyll b embeddedin thylakoid membranes, and a rich panoplyof accessory pigments developed to capturelight and protect the photosynthetic appara-tus from the unfiltered terrestrial light (80).

Generally, primary plastids have under-gone major modification during their tenurein the eukaryotic host; reduction of theirgenome’s coding capacity is one of the moreconspicuous attenuations. The genome ofthe cyanobacterium Anabaena sp. PCC 7120has 5366 protein-encoding genes, and othercyanobacteria possess similar numbers ofgenes (53). In contrast, the most gene-richplastid reported to date, that of the red algaPorphyra purpurea, encodes a paltry 251 genes(99), and the plastids of the parasitic plantEpifagus virginiana harbor a mere 42 genes(132). Thus, most of the original genetic ma-terial of the endosymbiont was clearly ei-ther lost or transferred to the host genomeduring their coevolution. Selection likely fa-vored the initial loss of genetic materialby the endosymbiont because it turned the

www.annualreviews.org • Plastid Evolution 493

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Serial2o

Karlodinium DinophysisKryptoperidinium

Lepidodinium

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Cyanothece

Cryptophyta(4, PB, Ca/c)

3o

3o3o

Dinophyta(3, Ca/c)

Perkinsidae(4)

Apicomplexa(4) Heterokontophyta

(4, Fcx, Ca/c)

Haptophyta(4, Fcx, Ca/c)

Euglenophyta(3, Ca/b)

Chlorarachniophyta(4, Ca/b)

Cyanobacterial ancestor(PB,Ca/b)

Glaucophyta(2, PB, Ca)

Oxyhirris Ciliata

Rhodophyta(2, PB, Ca/d)

Embryophyta(2, Ca/b)

2o

2o

2o

1o

Paulinella chromatophora(2, PB, Ca)

Chlorophyta(2, Ca/b)

1o

1o

Peridinium Karenia

Figure 1A schematic representation of plastid evolution. Engulfment of a cyanobacterial ancestor and subsequentreduction to a primary plastid (1◦) by a eukaryotic host initially led to the formation of three lineageswith primary plastids: the chlorophytes, and land plants, rhodophytes and glaucophytes. The subsequentuptake of a green or a red alga by independent hosts to form secondary endosymbioses (2◦) resulted ineuglenophytes, chlorarachniophytes, and the monophyletic chromalveolates. Chromalveolates, whichrepresent the association of chromists (Heterokontophyta, Haptophyta, and Cryptophyta) and theAlveolata (Apicomplexa, Perkinsidae, Dinophyta, Ciliata), unite an extremely diverse array of protists andnot all authors accept the grouping. Different Dinophyta have replaced their original secondary plastidwith a green alga either by serial secondary endosymbiosis (Lepidodinium) or even tertiary endosymbioses(3◦); e.g., Karlodinium harbors a tertiary plastid of haptophyte origin. The heterokontophyte Rhopalodiagibba engulfed a cyanobacterial Cyanothece species and reduced it to so-called spheroid bodies, which arenot used for photosynthesis, but rather act in N2-fixation. The plastid organelles were apparently lost inthe case of the ciliates and the dinoflagellate Oxyhirris. A possible nascent primary endosymbiosis (1◦) isrepresented by Paulinella chromatophora, although whether this endosymbiont is a true plastid organelleremains uncertain. The number of membranes surrounding the plastid and the photosynthetic pigmentsis shown in parentheses. PB, phycobilin proteins; Fcx, fucoxanthin; Ca/b/c/d, chlorophyll a/b/c/d.


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prokaryote-eukaryote consortium into an ob-ligate symbiotic relationship. However, wenow know that a concerted and ongoing trans-fer of genes from endosymbiont to host hasradically depleted the endosymbiont’s genecatalog. Much of this intracellular gene trans-fer was likely achieved prior to the divergenceof the three primary endosymbiotic lineagesbecause they share a similar residue of com-mon genes (75).

This transfer of genetic material mandatedthe development of a mechanism to returnthe gene product to the organelle. We dis-cuss this problem in detail below, but somegeneral concepts can be outlined now. Host-encoded proteins destined for the plastid aretypically translated as precursor proteins bear-ing an N-terminal topogenic signal that is rec-ognized by a proteinaceous receptor, whichis either soluble in the cytoplasm or boundto the outer plastid membrane. After recog-nition, the precursor is subsequently translo-cated across the plastid envelope by a suiteof translocation machineries spanning thetwo bounding membranes. The preprotein ispulled into the plastid and the topogenic sig-nal is proteolytically removed to yield the ma-ture protein.

The protein import mechanism probablyevolved early on in the conversion of the en-dosymbiont into an organelle and no doubtfacilitated the relocation of genes from theendosymbiont to the host. Transferred geneswould need to acquire expression and to-pogenic signals for the gene product to bereturned to the organelle. Rhodophtye andgreen algal/plant plastid-protein-targetingmachineries appear to be fairly similar (79);although virtually nothing is known about thetranslocation machinery in glaucophytes, wepredict that it is also similar because theseplastids have also relinquished so many oftheir genes to the host genome (75).

Eukaryotic Endosymbiosis

The current consensus of molecular phy-logeny recognizes six eukaryotic super-

Nucleomorph: theformer nucleus ofthe eukaryoticendosymbiont; lostin most secondaryalgae, but stillpresent in a highlyreduced form incryptophytes andchlorarachniophytes

clusters: Opisthokonta, Amoebozoa, Plantae(Archaeplastida), Chromalveolata, Rhizaria,and Excavata (2, 54). The Plantae su-percluster embraces the three lines (glau-cophytes, rhodophytes, and chlorophytes)with primary endosymbiotic (two membrane)plastids and their monophyly is consis-tent with a common origin for their plas-tids (103). However, plastids also occur inthe Chromalveolata, Rhizaria, and Exca-vata, and all these multi-membrane plastidsare derived from secondary endosymbioses(Figure 1). These events created a varietyof eukaryotic-eukaryotic chimeras referred toas meta-algae (22). Secondary or complexplastids are derived from eukaryotic, primaryplastid-containing endosymbionts and haveundergone reduction during their tenure inthe secondary host. The degree of reductionvaries; sometimes it is relatively minor, suchas in the partially integrated secondary en-dosymbionts of Hatena (85), and sometimesit is extensive, such as in the case of eu-glenoids in the Excavata where the only traceof the eukaryotic endosymbiont is an extra(third) membrane around the plastid (130).Two important intermediate stages in thesecondary endosymbiont reduction processare represented by cryptophytes and chlo-rarachniophytes, in which a very reduced en-dosymbiont nucleus, cytoplasm, and plasmamembrane can still be identified. The rem-nant nucleus, known as the nucleomorph,is located inside the periplastidial compart-ment (the former endosymbiont’s cytosol),and the overall topology allows us to rational-ize the presence of four membranes aroundrelated plastids in chromalveolates, in whichthe endosymbiont nucleus has completelydisappeared. Reduction forces have obviouslybeen at work in these endosymbionts becausethe great majority of the endosymbiont nu-clear genes have been transferred to the hostnucleus and most of the cytoplasmatic fea-tures, other than a small collection of ribo-somes, have been lost (27, 33).

Secondary endosymbioses introducedplastids into heterotrophic lineages, and


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Chromalveolatehypothesis:monophyly of thechromists(Cryptophyta,Haptophyta andHeterokontophyta)and Alveolata(Dinophyta, Ciliataand Apicomplexa);also, the commonancestor contained acomplex plastidderived from a redalga that is retainedin several of theselineages

much energy has been focused on establish-ing how many separate times a eukaryoticsymbiont has been integrated into a pre-viously nonphotosynthetic lineage. Theenvironmental and commercial importanceof the lineages created, not to mention theimportance of these events as drivers ofeukaryotic diversity, make this a particularlyfascinating question. The antiquity of theseevents and the reduction processes thathave occurred in the ensuing millennia alsomake the question a difficult one to re-solve. The most parsimonious hypothesis,put forward by Cavalier-Smith, invokesonly two secondary endosymbioses: oneinvolving a green alga leading to theCabozoa (which unites euglenophytes andchlorarachniophytes) and one involving ared alga that created the Chromalveolata(which unites cryptophytes, haptophytes,heterokontophytes, dinoflagellates, perkin-sids, apicomplexa, and the plastid-lackingciliates) (22). Interestingly, no examplesof a glaucophyte secondary endosymbiontare known. Various lines of evidence now

refute the Cabozoa hypothesis (7, 10, 33, 66,104) and it is now clear that two separateacquisitions of green algal endosymbiontscreated the euglenophytes and chlorarach-niophytes independently. The veracity ofthe chromalveolate hypothesis remainsuncertain, and whether or not the chrom-alveolates are derived from a single or multi-ple secondary endosymbioses of separate redalgal endosymbionts is still much debated.The chromalveolate hypothesis finds somesupport from molecular phylogenies (44, 86),and some unusual recruitments of enzymes tothe endosymbiont shared by chromalveolatesalso lend credence to a single secondaryendosymbiotic event (9, 30, 43, 44, 88). Itwas argued early on that the mechanism ofhow proteins are targeted from the host tothe complex plastid would give insight intothe endosymbiont’s ancestry (22), and recentinsights into this process (see Figure 2 andbelow) are congruent with the chromalveo-late scenario. Drawn together, these differentanalyses support the idea of a monophyleticorigin for chromalveolates from a single red

−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−→Figure 2Models of the machineries that import nuclear-encoded plastid proteins for select primary and secondaryplastids. Nuclear-encoded factors are brown, plastid-encoded factors are green, and nucleomorph-encoded factors are gray. Organisms with primary plastids (green algae, plants, and rhodophytes) sharecore components of the import apparatus, although land plants apparently have a more elaborate set ofreceptors (Toc159 and Toc64) in the outer envelope membrane (OEM) and other participating factors(Tic55 and Tic40) in the inner envelope membrane (IEM). These factors, together with Tic62, might beinvolved in redox-regulated import. Oep16 imports protochlorophyllide oxidoreductase A from thecytosol in Arabidopsis independently of the canonical Toc system. A more complicated import pathway isnecessary for secondary plastids, as in the case of the cryptophytes, which are surrounded by additionalmembranes, namely the periplastidial membrane (PPM) and rough endoplasmatic reticulum (rER). Incryptophytes preproteins are cotranslationally inserted into the ER via the Sec61 complex and the signalpeptide (SP) is cleaved by the lumenal signal peptide peptidase (SiPP). The remaining transit peptide(TP) mediates translocation across the remaining three membranes, before being cleaved by the stromalprocessing peptidase (StPP) inside the stroma, similar to primary plastids. Whether the secondary plastidof P. falciparum and chlorarachniophytes is actually located within part of the ER, as in cryptophytes andother chromists, is uncertain. Morphology obviously has a significant impact on the actual importpathway and machinery necessary. Proposed models for complex plastids are mostly inferred fromgenome data mining and lack experimental proof. Tic20 and Der1-2 have not yet been identified incryptophytes (question marks) but genes that encode proteins believed to be targeted into the plastid arepresent in other chromalveolates for which full genome sequence is available. PPC, periplastidialcompartment; EPM, epiplastid membrane. Topogenic signals for stromal targeting are displayed beneaththe organisms’ names. The F-motif, which is apparently critical for stromal targeting, occurs in plastidswith red algal origin.


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Eukaryoticendosymbiosis: allevents in which theengulfed organismthat was reduced toan organelle was aeukaryote

Endosymbiontmetabolicreplacement:replacement of anexisting host-cellmetabolic pathwaywith one acquiredwith theendosymbiont

alga endosymbiont. However, some analyseswith genes encoding cytosolic host proteinsdo not support the chromalveolate hypothesis(86, 120), suggesting that the spread of asingle red algal endosymbiont among thechromalveolate branch may have occurred bysubsequent tertiary endosymbioses. Further-more, the clustering of genes representingRhizaria together with the Chromalveolatain a recent report by Hackett and colleagues(40) reminds us that definite proof for themonophyletic origin of chromalveolates hasnot been found.

One further aspect of eukaryotic endosym-biosis is tertiary, maybe even quaternary, andserial secondary endosymbiosis. Tertiary en-dosymbiosis is the uptake of a secondaryendosymbiosis-derived alga by a eukaryote,and serial secondary endosymbiosis is the re-placement of an original complex plastid witha new, primary endosymbiosis–derived alga.Select dinoflagellate algal lineages representthe best-studied cases of these higher orderendosymbiotic events, and independent casesare represented by the genera Lepidodinium,Kryptoperidinium, Karlodinium, and Dinophysis(for detailed description of these unusual di-noflagellates lineages see References 39, 46,and 54) (Figure 1). In each case, the host di-noflagellate previously contained a secondaryplastid, so these new endosymbionts representorganelle replacements. The mechanisms fororganelle reduction and integration are likelythe same for secondary endosymbionts; how-ever, in cases of organelle replacement eventransferred genes from the first plastid cancontribute to the integration of these new re-cruits (49, 91).

Nature’s Playground:The Evolution Continues

Plastid loss and reversion to obligateheterotrophy. A fascinating but often over-looked element of endosymbiotic theory con-cerns organelle reduction and loss. In a sense,all endosymbiotic organelles are products ofmassive reduction of the metabolic complex-

ity and capabilities of the ancestral free-livingsymbiont. But there is a tendency to regardfunctional organelles as having reached a sta-ble suite of core metabolic functions—in thecase of plastids photosynthesis is consideredthe cornerstone of organellar function (seebelow for summary of plastid biochemicalfunctions). Despite this mindset, an extensivenumber of lineages have independently ad-vanced their plastids a further rung on theladder of reduction by losing their photo-synthetic capability (21). Parasitic plants andapicomplexan parasites such as the malariaparasites are notable examples; many otherprotists have also lost the ability to per-form photosynthesis but retained their fur-ther reduced plastids (e.g., the euglenid As-tasia and the dinoflagellate Crypthecodinium).These nonphotosynthetic plastids apparentlystill provide essential services to the hostcells—for instance, fatty acid synthesis, iso-prenoid synthesis, and heme synthesis in thecase of the malaria parasites (96). Most ofthese additional plastid pathways have likelyreplaced equivalent host cell pathways thatoccurred in the ancestral host cell prior toplastid acquisition (endosymbiont metabolicreplacement). Why a plastid pathway shouldreplace an existing host cell pathway is un-clear, although it is quite conceivable thatchance has played a role in the eliminationof any one of the duplicated pathways afterendosymbiotic merger. In any case, fixationof the plastid copy of any essential metabolicpathway would commit a cell to plastid reten-tion even if photosynthesis was subsequentlyabandoned. What, then, is the likelihood ofsuch a cell achieving complete plastid loss?

To date, members of at least six ma-jor eukaryotic lineages may have achievedoutright plastid loss: ciliates, the apicom-plexan (e.g., Cryptosporidium), dinoflagellates(several lineages), heterokontophytes (e.g.,oomycetes), trypanosomatids, and crypto-phytes (Goniomonas). However, for severalof these lineages such claims have inspiredlively debate. The case for plastid loss inalveolates (i.e., ciliates, apicomplexans, and


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dinoflagellates) largely hinges on acceptanceof the chromalveolata hypothesis, whichunites alveolates with chromists (heterokon-tophytes, cryptophytes, and haptophytes) andproposes plastid origin in a common ancestor(22). If this hypothesis is correct, ciliates andbasal lineages of apicomplexans (Cryptosporid-ium and gregarines) and dinoflagellates (e.g.,Oxyrrhis, Amoebophyra, Noctiluca) that all lackplastids must have independently lost theseorganelles (1, 107, 121, 134). Challenging thisscenario is the lack of strong phylogeneticevidence for the monophyly of chromalveo-lates host cells (40, 86, 120). Thus, an alter-native explanation for plastid occurrence inalveolates is that apicomplexans and dinoflag-ellates independently gained their plastids,and that ciliates and the basal members of eachancestrally lacked a plastid. The dinoflagel-late lineage, however, may represent an in-dependent case for plastid loss, because sev-eral nonphotosynthetic groups are apparentlyscattered throughout photosynthetic di-noflagellates [according to small subunitrRNA phylogenies and plate tabulation data(107)], implying several independent losses.However, loss of photosynthesis may notalways imply plastid loss, and the recentdemonstration that at least one such taxon(Crypthecodinium) retains a nonphotosyntheticplastid indicates that plastid loss should bemore closely examined in this group (108).

In contrast to the conspicuous photo-synthetic members of the heterokontophytessuch as kelp and diatoms, many mem-bers (e.g., thraustochytrids or oomycetes) arenonphotosynthetic (23). Oomycetes are wellknown plant pathogens, responsible for sig-nificant historical events such as the Irishpotato famine. Most nonphotosynthetic het-erokontophytes fall into basal clades (23), andtherefore again the question of plastid losshinges on whether heterokontophytes sharea common plastid with other major lineages(i.e., other chromists, or indeed all chromalve-olates) or whether a plastid was independentlyacquired within the heterokontophyte radi-ation. Trypanosomatids are a heterotrophic

group of parasites whose sister relationship tothe euglenids (many of which are photosyn-thetic) has inspired suggestion that these par-asites also once contained a plastid but havesince lost it (41). However strong evidence forsecondary plastids as a recent gain in the eu-glenoid lineages (66), along with the rebuttalof the Cabozoa hypothesis (see above), under-mines the case for plastid loss in trypanoso-matids.

Perhaps the strongest case for plastid lossoccurs in the Cryptophyta. Most cryptophytesare photosynthetic, although some have ap-parently lost photosynthesis but retain a relictplastid (113). Conversely, Goniomonas is abasal heterotrophic cryptophyte that appar-ently lacks a plastid (77). Recent phylogeniesbased on molecular data have strongly identi-fied haptophytes as the sister lineage to cryp-tophytes (40, 86), and a gene replacement ofplastid-encoded rpl36 is uniquely shared bythese taxa, implying they share a commonplastid (55, 101). Thus, reasonable evidenceexists that the common ancestor of crypto-phytes and haptophytes contained a plastid,and therefore Goniomonas has lost its plastid.

Although further cases of plastid losswill likely be substantiated as global phy-logenies develop better resolution, a casefor plastid loss in cryptophytes at leastlooks well supported. How then, is a eu-karyote able to reverse the endosymbioticprocess—particularly, how can endosymbiontmetabolic replacement be reversed? Two sce-narios are possible. One is that the plastid islost relatively early in endosymbiont integra-tion, before endosymbiont metabolic replace-ment occurs. Cavalier-Smith (19) has sug-gested that this accounts for why plastidlesstaxa are often basal to photosynthetic lineages;they represent the period before a cell startsto rationalize its own biochemistry and relyon elements of the symbiont’s biochemistry. Ifthe chromalveolate hypothesis is correct, thenonly after the major lineages diverged did theplastid become essential beyond photosynthe-sis, because most of the major lineages haveplastidless basal members. A second scenario


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is that through heterotrophy a cell can satisfyits requirement for the macromolecules it hadcome to rely upon from the plastid. Many sec-ondarily nonphotosynthetic groups are eitherpredators or highly adapted parasites, with ac-cess to a rich supply of macromolecules. Ifthis diet can satisfy the need for fatty acids,isoprenoids, and heme, for instance, a cellmight be well on the way to making its plastidobsolete.

In either case, if a plastid is lost from a eu-karyotic lineage, would evidence of the plas-tid’s tenancy remain? The many hundreds tothousands of nucleus-encoded plastid geneswould initially be present, but without a func-tion these would likely degrade quite rapidly.However, any plastid genes that had cometo fulfill an alternative, nonplastid-localizedfunction would remain useful even after plas-tid loss. Such genes, referred to as EGT(endosymbiont gene transfers) have been esti-mated to represent between 10% of nucleus-encoded genes derived from the plastid inglaucophytes and 50% in plants (73, 100).This amounts to ∼150 and ∼2250 genes in to-tal in the two groups, respectively. Thus, lossof a plastid could conceivably leave a conspic-uous footprint if these genes remained usefulin the absence of the plastid. In the oomycetesone such plastid-derived gene ( gnd ) has beenhailed as evidence for the former plastid in thislineage (6). However, to form a compellingcase for a former plastid we should antici-pate a large collection of such genes whenthe annotation of oomycete genomes is com-plete. Conversely, ciliates have revealed nosuch plastid footprint; the complete sequenceof Tetrahymena thermophila was recently inves-tigated for just such relict plastid genes (29). Itwill be very interesting to undertake the sameanalysis for Goniomonas in pursuit of a betterunderstanding of the process of plastid loss.

Symbioses in progress. In addition to full-fledged plastid organelles, numerous organ-isms demonstrate that endosymbiosis is a con-tinual driving force in evolution. Here wepresent four interesting cases of organisms at

various points of negotiation of these cellularmarriage contracts.

Rhopalodia gibba is a diatom that hostsboth a secondary red algal–derived plas-tid and a novel cyanobacterial symbiont(Figure 1) known as the spheroid body (32).Unlike cyanobacterial-derived plastids, whichare typically photosynthetic, the spheroidbody has apparently lost this ability. However,the spheroid body has retained another corecyanobacterial function, the ability to fix ni-trogen, which it performs for its diatom hostduring the day (93). Spheroid bodies are in-herited vertically from one generation to thenext, and their numbers are regulated in thehost cell, which implies a high level of hostcontrol over the endosymbiont. As yet no ev-idence for spheroid body genes in the diatomhost has been found, so whether the final stageof organelle integration has been achieved inthis case remains undetermined. However, thespheroid bodies have clearly suffered geneloss, and they are most likely incapable ofagain living outside the host (93). Nitrogenfixation is another of the innovations specificto prokaryotes, so it is noteworthy that en-dosymbiotic capture again has played a rolein extending such fundamental capabilities toeukaryotes.

A further independent example of pri-mary endosymbiosis is seen in the freshwa-ter amoeba Paulinella chromatophora, whichalso hosts a cyanobacterium-like symbiont(56). In this case photosynthesis is retained,and P. chromatophora accordingly has con-verted from heterotrophy to autotrophy. ThePaulinella endosymbiont, referred to as acyanelle, occurs in the cytoplasm without anyadditional bounding membranes, and sym-biont numbers are strictly regulated, againsuggesting a higher level of host-symbiontinteraction (133). Attempts to culture thesymbiont separate from the host have thusfar been unsuccessful, but analysis of thePaulinella cyanelle genome reveals neither ob-vious gene loss nor transfer of genes to thehost, so it appears that there has been rela-tively little genetic response to this union so


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far (133). The Paulinella symbionts are thusenigmatic; however, these symbionts poten-tially represent a second case of primary en-dosymbiosis enabling photosynthetic capture.Fortunately, Paulinella chromatophora hassymbiont-lacking sister species (P. indentataand P. ovalis), which offers the possibility ofunderstanding how this organism can acquirea permanent prokaryotic endosymbiont in aprocess reminiscent of the origin of plastids.

Nascent secondary endosymbioses arealso in evidence. The enigmatic flagellateHatena arenicola harbors a quasi-permanentprasinophyte-like endosymbiont (related tothe genus Nephroselmis), which exhibits sub-stantial structural modification when withinthe host. Permanent integration of this pho-tosynthetic symbiont is apparently pendingbecause division of the symbiont is not yetcoordinated with that of the host (85). Never-theless, a degree of integration has apparentlyoccurred, because host cell division resultsin one daughter cell inheriting the endosym-biont while the other daughter cell is left with-out a symbiont and presumably sources a newsymbiont from the environment.

Perhaps one of the more startling cases ofendosymbiosis in progress is the plastid theftperformed by the sea slug Elysia chlorotica.This sea slug feeds upon algae and can sal-vage the plastids from their diet of Vaucherialitorea and maintain them, generating pho-tosynthate that can nourish the animal formany months. The plastids, which are arrayedin specially generated diverticulae of the sluggut to be exposed to incoming light, remaintranscriptionally and translationally active forup to nine months (83). Circumstantial evi-dence suggests that the plastids even receiveproteins synthesized by the sea slug (42). Ifsubstantiated, this would implicate horizontaltransfer of a gene from the alga to the sea slug,which by one definition would make this cap-tured plastid an animal organelle. However,these plastids are unable to divide, and arenot passed on from one slug generation to an-other, nor do they occur in the animal’s germline. In fact, stolen plastids such as these could

probably never achieve permanent endosym-biont status because many essential plastidgenes would have been left behind when thealgal nucleus was digested. Sea slug plastidsare thus examples of kleptoplastids: photosyn-thetic organelles stolen from another organ-ism but not permanently acquired.

PREPROTEIN TARGETING

Intracellular gene relocation is dependent onthe existence of a system to reimport thegene product back to the compartment oforigin. Given the massive scale of plastid-to-nucleus gene relocation, this system mustrecognize and sort a large number of plastid-destined proteins from all other proteins syn-thesized in the cytoplasm. Elements of thissystem, including proteins of the import ap-paratus embedded in the plastid membranesand some features of plastid precursor pro-teins, are shared throughout phototrophiceukaryotes and reflect the common originof primary plastids. Core elements of thesystem shared by red algae and green al-gae/plants clearly arose early, prior to the di-versification of the primary endosymbiont-containing lineage (79). In plants additionalelements such as extra receptors and redox-sensing components of plastid protein importclearly have arisen to optimize this systemand also facilitate the biogenesis of differentplastid types (e.g., amyloplasts, chromoplasts,and chloroplasts) (14). Great insight has nowbeen achieved into the complex and sophis-ticated plastid protein import machinery ofplants, the details of which are reviewed else-where (14, 38, 115). Here, we confine our-selves to a synopsis of the primary plastidimport system and give more focus to theless well-understood machinery for proteinimport into secondary plastids with multiplebounding membranes.

Targeting to Primary Plastids

Primary plastids contain three distinct setsof membrane: the outer envelope membrane


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(OEM), the inner envelope membrane (IEM),and the thylakoid membranes, which thuscreate three separate compartments (inter-membrane space, stroma, and thylakoid lu-men). Proteins can therefore be targeted tosix regions within plastids: three membranesand three soluble compartments. Dedicatedtranslocation machineries and peptide target-ing information within the nuclear-encodedplastid proteins are used in concert to achievethese targeting feats (Figure 2).

The majority of proteins is targeted tothe plastid posttranslationally, facilitated by anN-terminal transit peptide extension. Thispeptide leader can vary in length from approx-imately 20 to 150 amino acids and no primarysequence consensus or common secondarystructure has been identified in the large col-lections of transit peptides known from dif-ferent plants. General characteristics includehydrophobicity at the extreme N terminus,enrichment of hydroxylated amino acids, anda depletion in acidic residues that leads toa positive charge, particularly toward the Nterminus (16, 89, 95). Some transit peptidesare phosphorylated by an ATP-dependent cy-tosolic kinase, which leads to an interactionwith a guidance complex; these peptides arethen preferred for import (76). After translo-cation, transit peptides are cleaved off by thestromal processing peptidase, which belongsto the M16 family of metallopeptidases, re-leasing the mature protein into the stroma(102).

Transport of the majority of preproteinsacross the two envelope membranes is thejob of the Toc (translocator of the outerchloroplast membrane) and Tic (transloca-tor of the inner chloroplast membrane) ma-chineries. These two apparatuses comprisemultiple soluble and membrane-bound pro-teins named for their molecular masses (seeFigure 2). Toc75 is the main translocationpore in the outer membrane (28) and togetherwith Toc 33/34 and 159 makes up the Toccore (111, 112). Other Toc components ap-parently have subsidiary roles; for example,Toc64 is implicated in plastid protein recog-

nition and delivery to the Toc pore. In plantssuch as Arabidopsis multigene families encodedifferent (partially redundant) isoforms of Toccomponents and differential isoform expres-sion probably generates import complexes tai-lored to particular plastid states in differenttissues (12, 51). Conversely, the haploid mossPhyscomitrella patens appears to lack these iso-forms, and thus is emerging as a superiormodel for gene knockout studies of Toc/Ticfunction (47). Interestingly, in the genomeof the red alga Cyanidioschyzon merolae, onlyToc34 and Toc75 have thus far been identi-fied (Figure 2), which might mean that majorreceptor components of the outer membrane(e.g., Toc 159 and 64) of land plant plastidsare specific to this green lineage, and that red-specific Toc receptors await discovery (79).

The core of the Tic complex includesTic20, Tic22, and Tic110 (Figure 2). Tic22is a soluble protein in the intermembranespace and is thought to be the first Tic com-ponent to interact with incoming precursors(59). Either or both of the two membrane pro-teins Tic20 and Tic110 could be involved inpore formation but details are unclear (50, 60,122). Tic110 also interacts with the chaper-one Hsp93 (ClpC) inside the stroma (3). Asimilar function is proposed for Tic40, be-cause it possesses a conserved domain knownfrom Hsp70 cochaperones (24). Several re-ports suggest the other Tic components ofland plants (Tic32, Tic55, and Tic62) mightbe involved in the redox-regulated import ofpreproteins (13, 61). The presence of ho-mologs for Tic20, Tic22, Tic62, and Tic110in the genome of the red alga C. merolae, com-bined with the apparent absence of Tic32 and55, might suggest that the former are essen-tial and the latter dispensable for functionalplastid import (79), but experimental confir-mation is needed. Finally, on the stromal sideof the membrane chaperones such as GroELand CplC interact with the Tic complex toreceive the imported proteins and fold them,after cleavage of the transit peptide, to theirmature conformation (125). However, someproteins require further targeting and the


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thylakoid membranes contain no less thanthree independent sets of protein transloca-tion machineries for this purpose: the sig-nal recognition particle–dependent (Albino3)pathway, the Tat (twin arginine translocon)pathway, and the Sec-dependent pathway. Inaddition, spontaneous insertion of proteinsinto thylakoid membranes is also known tooccur, thus offering at least four alterna-tive routes into the membrane or lumen ofthylakoids (38).

Alternative, Toc/Tic-independent routesto plastids are also becoming apparent (60,97). For instance, the outer envelope pro-tein 16 (Oep16)—a homolog of bacterialpreprotein and amino acid transporters—serves as the translocase for one plastid pro-tein, NADPH:protochlorophyllide oxdidore-ductase A (98). Another noncanonical importpathway through the outer envelope mem-brane was recently revealed with the identi-fication of nuclear-encoded plastid proteinspossessing N-terminal signal peptides ratherthan the standard plastid transit peptides (94,126). These plastid proteins traverse the en-doplasmic reticulum (ER), and most likelyalso traverse the Golgi apparatus where theyare glycosylated, and are subsequently tar-geted to the outer envelope membrane of theplastid (94, 126). The details of this alternateroute remain mysterious.

Targeting Into and WithinSecondary Plastids

Translocation of precursor proteins to sec-ondary plastids must surmount additionalobstacles in the form of extra bounding mem-branes, which also creates additional compart-ments that have their own specific proteomes.Three membranes surround dinoflagellateand euglenophyte complex plastids, whereascryptophyte, heterokontophyte, haptophytes,apicomplexan, and chlorarachniophyte plas-tids are surrounded by four membranes (22).Independent origins of secondary plastidshave resulted in distinct targeting solutionsto these advanced trafficking needs; however,

Epiplastidmembrane (EPM):the outermostmembranesurrounding complexplastids

remarkably, some unifying principals haveemerged. Virtually all known complex plastidpreproteins encoded in the nucleus possess anN-terminal topogenic signal composed of atleast two parts: a signal peptide and a transitpeptide. The ubiquity of what appears to bea canonical signal peptide in complex plastid-targeted proteins is consistent with the out-ermost membrane being a component of thehost cell’s endomembrane system, apparentlyderived from the formative phagocytic event(22). The signal peptide mediates cotransla-tional import into the ER lumen, where signalpeptidase removes the signal peptide to exposethe transit peptide, which is responsible fortargeting across the remaining membranes.An unusual elaboration of this bipartite leaderoccurs in the two cases of complex plastids sur-rounded by three membranes: dinoflagellatesand euglenoids. Here an additional signal, ahydrophobic membrane anchor, is embeddedin the transit peptide region of most plastidproteins (84, 87, 118). Thus, insertion of pre-proteins into the ER lumen in these taxa isapparently delayed; plastid preproteins are an-chored to endomembranes until they are de-livered to the plastid and the complete plastidimport is enabled.

In heterokonts, haptophytes, and crypto-phytes the outer plastid membrane is con-tinuous with the host rough ER and thusis studded with ribosomes (18). Plastid pro-teins encoded in the nucleus have thereforealready passed through the first of four mem-branes after cotranslational insertion into theER lumen by the N-terminal signal peptide(Figure 2). However, in other complex plas-tid systems no such continuity of plastids andER is apparent, so plastid preproteins mustbe delivered from the ER lumen to the outerplastid membranes [termed epiplastid mem-branes (EPM) in Figure 2], presumably byvesicular traffic (84, 118). After signal pep-tide cleavage, plastid proteins must be dis-tinguished from secretory proteins; mutage-nesis experiments in several complex plastidsystems indicate that the transit peptide is re-sponsible for this discrimination (34, 62, 129).


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Periplastidialcompartment(PPC): the reducedcytosol of theengulfed alga;harbors thenucleomorph and80S ribosomes incryptophytes andchlorarachniophytes

Periplastidialmembrane (PPM):represents theformer cytoplasmicmembrane of theendosymbiont and isthe secondoutermostmembranein four-membrane-bounded plastids

The signal peptide and transit peptide thusact sequentially to mediate targeting into thestroma of complex plastids.

At least for some complex plastids, proteinsare also targeted into the periplastidial com-partment (PPC); these proteins also utilize abipartite leader. The most N-terminal residueof the transit peptide is critical in dictatingwhether a protein travels all the way throughthe three innermost plastid membranes to thestroma or whether it stops in the PPC aftertraversing only the periplastidial membrane(35, 116). In the cryptophyte Guillardia thetaand the heterokontophyte Phaeodactylum tri-cornutum, this +1 transit peptide residue istypically a phenylalanine for stromal proteins(in a few exceptions other aromatic aminoacids fulfill this role). In the absence of thisphenylalanine the preprotein accumulates inthe PPC (34, 37). In other chromalveolates anaromatic amino acid–based motif (F-motif) isalso a conspicuous feature of the N terminusof transit peptides, and likely plays a role incorrect stromal targeting (35, 58, 90). Inter-estingly, this F-motif also occurs in the transitpeptides of plastid-targeted proteins of glau-cophytes and rhodophytes (117), which sug-gests that the F-motif could be an ancient tar-geting element for plastid import. Thus, incomplex plastids the role of this F-motif hasapparently been extended to discriminate be-tween proteins that are required to be targetedfully into the plastid stroma and those thatmust be halted in the PPC. The corollary isthat the remainder of the transit peptide is suf-ficient for targeting across the periplastidialmembrane (PPM) (Figure 2). Curiously, theF-motif does not occur in transit peptides ofthe green algal/plant lineage, and thus thisancient targeting signal has apparently beenabandoned here. This might explain the ap-parent need for extra receptors like Toc159and 64 in these plastids (Figure 2).

The second step of protein trafficking intocomplex plastids (the translocation from theER lumen into the PPC, see Figure 2) pre-sumably requires a translocon in the PPM.Termed the Top translocon (22), this hy-

pothetical membrane transporter and its in-triguing evolutionary pedigree may recentlyhave been identified. The nucleomorph ofthe cryptophyte G. theta encodes ERAD (ER-associated degradation) components, includ-ing a Der1p (degradation in the ER) mem-brane translocon able to complement ERAD-deficient yeast (116). Because no ER is presentinside the periplastidial space, the location ofthis nucleomorph-encoded ERAD machin-ery was intriguing. Preliminary immunolo-calization studies suggest that the Der1ptranslocon is located in the PPM of the cryp-tophyte’s complex plastid (116), leading tospeculation that it could be the long sought af-ter Top translocon. Further credence for thishypothesis comes from the identification ofan extra set of ERAD machinery (distinct fromthe canonical host ER ERAD machinery) thatis apparently targeted to the complex plas-tids of other chromalveolates such as diatomsand Plasmodium (116). Because these plastidshave lost all traces of the endosymbiont cy-toplasm it is highly plausible that this ERADmachinery could localize to the periplastidialmembrane and have a role in translocatingtransit peptide–bearing preproteins intothe complex plastids. The ERAD-derivedtranslocon is proposed to recognize the transitpeptide (which might resemble an unfoldedprotein similar to the normal ERAD sub-strate) and to pull the precursor proteins outof the ER and into the periplastidial compart-ment (Figure 2).

Although the role of this ERAD appara-tus in targeting proteins to complex plastidsremains to be substantiated, it provides tan-talizing support for the chromalveolate hy-pothesis. As alluded to above, solving the“protein-import problem” was a major hurdlein the establishment of secondary endosym-bionts (18). The apparent co-option of a nor-mally ER-based protein translocation systeminto plastid transport by cryptophytes, het-erokontophytes, and Apicomplexa is sugges-tive of a common origin for the plastids.Similarly, use of the F-motif to discriminatebetween periplastidial and stromal-directed


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proteins by cryptophytes, diatoms, and per-haps even apicomplexa is also congruent witha common origin for their plastids.

BIOCHEMICAL PATHWAYS

The union of a heterotroph and an au-totroph in an endosymbiotic partnershipamalgamates two suites of metabolic path-ways into a single organism (131). The driverfor the union is typically believed to be theacquisition of photosynthesis by the host.Thus, both primary and secondary endosym-bioses likely converted heterotrophs into pho-totrophs. Some serial secondary endosym-bioses and tertiary endosymbioses may havesimply exchanged one photoautotrophic en-dosymbiont for another, but in general wecan frame the question in terms of het-erotroph + autotroph = new autotroph.From a metabolic perspective this fusion cre-ates interesting possibilities. Autotrophs aretypically self-sufficient metabolically; somerequire vitamins, but in general they synthe-size everything they need from scratch. Con-versely, heterotrophs have access to a rangeof preformed macromolecules in their dietand are able to salvage various building blocksfrom these macromolecules and utilize themin their metabolism. Thus, as a general princi-ple, the endosymbiosis likely introduced extrametabolic capability beyond just photosyn-thesis to the host’s repertoire. A key challengeis to unravel which pathways were introducedinto the amalgam from the endosymbiont. Asdiscussed above, the host can become depen-dent on endosymbiont pathways other thanphotosynthesis, and this dependency can im-pact plastid persistence should the organismsubsequently revert to a totally heterotrophiclifestyle.

What do we know about the metabolicrepertoires of the original hosts and endosym-bionts? For the hosts we can say very little. Wehave a relatively poor understanding of thenature of the host for the primary endosym-biosis, and, similarly, we are largely ignorantof the host’s affinities for the three known sec-

ondary endosymbioses (euglenophytes, chlo-rarachniophytes, and chromalveolates). It isthus rather difficult to speculate on what kindof metabolisms these hosts could have had atthe outset of the endosymbiotic relationship.However, we are in a better position to hy-pothesize about the metabolic repertoire ofthe endosymbionts. For primary endosymbio-sis we can postulate that the endosymbionthad a suite of metabolic capabilities similar tothose in modern-day cyanobacteria. For sec-ondary endosymbioses we can assume that theendosymbionts had a metabolic potential sim-ilar to that in the modern representatives ofred or green algae as appropriate.

Weeden (131) was the first to ponder froma metabolic perspective the consequences offusing an endosymbiont and host. He recog-nized that the endosymbiont introduced novelpathways into the host and he outlined howamino acid, heme, and starch pathways wereinducted into hosts via endosymbiosis. Wehave subsequently learned that cells have alsoexercised considerable creativity during thesemetabolic mergers, and complex amalgams ofhost and symbiont pathways have also beenthe fruits of these partnerships.

Starch Synthesis

Excess photosynthate is generally stored asglucan polymers. Plants and green algae storestarch (α-1,4 glucan) in the plastid, whereasred algae store starch in the cytosol (127). Onthe basis of these localities of starch synthe-sis, red algae were assumed to utilize a host-derived glucan synthesis mechanism whereasthe green algae and plants were assumed toemploy a system derived from the endosym-biotic ancestor of the plastid (Figure 3). Thestarting points for each of these pathways—UDP-glucose precursors for red algae andADP-glucose precursors for green algae andplants—also reflect the dichotomy betweeneukaryotic and prokaryotic glucan pathways.However, in reality both host and symbiontproteins have been recruited in starch syn-thesis in both red and green algae, and only


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UDP-G

Florideanstarch

ADP-G

GlycogenGlycogen

UDP-GADP-G

Starch

Rhodophyta Chlorophyta

Nuc

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Figure 3Schematic representation of starch synthesis before and after primaryendosymbiosis. Nuc, nucleus; UDP-G, uridine-diphosphate glucose;ADP-G, adenosine-diphosphate glucose.

the localities, either cytosolic or organellar,have been derived from either host or sym-biont (87). Why red algae retained the site ofhost glucan storage whereas green algae (andtheir descendants) adopted the endosymbiontstorage site remains unknown (Figure 4).

In secondary endosymbiosis glucan stor-age distribution differs: Sometimes it is in the

Nuc

MevPyruvate(DOXP)

IPPIPP

Nuc

Pyruvate(DOXP)

Mev

IPP IPP

Figure 4Schematic representation of the isopentenyl diphosphate (IPP) synthesispathway before and after primary endosymbiosis. Mev, mevalonate;DOXP, 1-deoxy-D-xylulose 5-phosphate.

host, and sometimes it is in the endosym-biont. For instance, euglenoids store paramy-lon (β-1,3 glucan) in the cytosol, althoughtheir endosymbiont is thought to have beena green alga that presumably stored starchin the plastid (26, 128). Chlorarachniophytesand heterokontophytes also store β-1,3 glu-cans in the secondary host cytosol and haveabandoned the glucan storage systems of thegreen and red algal endosymbionts, respec-tively (78). Conversely, cryptophytes storestarch in the PPC (remnant endosymbiont cy-toplasm), thus conserving the endosymbiontglycan storage system of the red algal en-dosymbiont (34). Dinoflagellates also storestarch; however, rather than in a PPC, storageoccurs in the host cytoplasm, implying reloca-tion of this pathway from the red algal cytosolto that of the host (25). Thus, the storage ofsurplus photosynthate in either the host or en-dosymbiont compartments has taken a rangeof alternatives in both primary and secondaryendosymbiotic partnerships.

Isopentenyl Diphosphate (IsoprenoidPrecursor) Synthesis

Isopentenyl diphosphate (IPP) is a buildingblock for terpenes, sterols, carotenoids, andisoprenoids that are important components ofa diverse range of cellular molecules such aschlorophylls and quinones. IPP synthesis wasonly recently discovered to occur in plastids(68). In plants the cytosol harbors the canoni-cal mevalonate pathway for IPP synthesis (8),and for many years it was presumed that thiswas the sole source of isoprenoid precursorsin plants. Given the extensive use of isoprenesubunits in plants for secondary metabo-lites such as terpenes, chlorophylls, ubiquinol,prenylated proteins, and isopentyl tRNAs, itis sobering to reflect that a plastid-based, non-mevalonate, deoxyxylulose (DOXP) pathwayfor IPP synthesis was overlooked, or at leastunrecognized, by plant physiologists. How-ever, once it emerged that bacteria synthe-size IPP from pyruvate and glyceraldehyde3-phosphate and not from mevalonate like


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eukaryotes, it was a simple step to identifya DOXP pathway in plastids (105). Indeed,the discovery of the plastid DOXP pathwayreconciled some previously incongruous pre-cursor incorporation and inhibitor data (67).Synthesis of IPP in plastids also simplifiesthe delivery of these entities to isopentenylatetRNAs for plastid translation and to isoprenechains for chlorophyll production (63).

In plants, IPP synthesis occurs in boththe host compartment (cytosol) and the en-dosymbiont compartment (plastid). The twodifferent pathways coexist and are even inte-grated to an extent (65), but their differencesare congruent with one (acetate/mevalonate)being derived from the host and the other(DOXP) being introduced with the cyanobac-terial symbiont (Figure 4). Exactly why bothpathways persist is not known but the require-ment for products in both the host and en-dosymbiont compartments perhaps necessi-tated the retention of two pathways.

Heme Synthesis

The synthesis of the tetrapyrroles that actas temporary electron carriers in various re-dox reactions is reminiscent of IPP synthe-sis in that there are two very different path-ways that begin with different substrates andutilize some, but not all, different enzymes.Many plastid-lacking eukaryotes utilize theso-called C4 or Shemin pathway, which com-mences by fusing succinyl-CoA and glycineto create δ-aminolevulinic acid (ALA), cour-tesy of aminolevulinic acid synthase (ALAS)(Figure 5). In animals and yeast, ALAS (114)is located in the mitochondrion and ALA isthen exported to the cytosol where a series ofsteps convert it to coproporphinobilogen III(CPIII). CPIII is then routed back into themitochondrion by a recently identified trans-porter for the last four steps to eventually pro-duce heme (Figure 5).

Cyanobacteria have a different initial sub-strate, commencing C-5–type heme synthe-sis from glutamyl-tRNA rather than succinyl-CoA and glycine (5) (Figure 5). Glu-tRNA

Nuc

Glycine+

Suc-CoA

Nuc

Glu-tRNA

Heme

Chl

Heme

Heme

Heme

Glu-tRNA

Heme

Chl

Figure 5Schematic representation of heme synthesis before and after primaryendosymbiosis. Nuc, nucleus; Glu tRNA, glutamyl tRNA; Suc-CoA,succinyl-Coenzyme A; Chl, chlorophyll.

reductase followed by Glu-SA aminomutaseconvert the aminoacylated tRNA to ALA.Steps from ALA to heme are then identical incyanobacteria and the Shemin pathway, andthe enzymes involved are homologous. How-ever, in cyanobacteria the pathway forks atprotoporphinobilogen IX. One branch leadsto heme as per the Shemin pathway, but theother branch involves the addition of Mg2+

to protoporphinobilogen IX to generatechlorophyll (124).

Because the original host for primaryendosymbiosis likely had aerobically respir-ing mitochondria, we can assume it had aShemin pathway to generate heme for itscytochromes. The acquisition of a cyanobac-terial endosymbiont almost certainly in-troduced the glutamyl-tRNA–based path-way into the first eukaryotic autotrophs. Atthe outset this organism would have hadtwo heme synthesis pathways: a Sheminpathway in the mitochondrion/cytosol anda cyanobacterial-like C5 pathway in theendosymbiont (Figure 5). To carry onphotosynthesis, the endosymbiont likelycontinued to synthesize chlorophyll fromglutamyl-tRNA; indeed, plant plastids stillsynthesize chlorophyll entirely within the


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plastid using a pathway homologous to that ofcyanobacteria (124). Interestingly, plants havedisposed of the early portions of the Sheminpathway and do not use glycine or ALASto commence heme synthesis (92). Rather,they export protoporphinobilogen IX fromthe plastid to the mitochondrion, which thenperforms the last two steps of heme synthe-sis using enzymes homologous to those ofthe animal/yeast Shemin pathway (Figure 5).It is noteworthy that plastids also completethe conversion of protoporphinobilogen IXto heme independently for the benefit of fur-nishing the prosthetic group for their own cy-tochromes.

For heme we thus see a picture of two path-ways rationalized into one pathway that forksthree ways: chlorophyll and heme synthesisfrom glutamyl-tRNA in the plastid, and hemesynthesis in the mitochondrion commencingnot from its original ALAS but from plastid-synthesised ALA (Figure 5).

Aromatic Amino Acid Synthesis

The essential amino acids are a necessary partof the animal diet because we lack a pathway

Mt

Nuc

Mt

Nuc

E-3-P+PEP

Phe

Trp

Tyr

Trp

E-3-P+PEP

Phe

Tyr

E-3-P+PEP

Phe

Trp

Tyr

Figure 6Schematic representation of aromatic amino acid synthesis before andafter primary endosymbiosis. Nuc, nucleus; Mt, mitochondrion; E-3-P,erythrose-3-phosphate; PEP, phosphoenolpyruvate; Tyr, tyrosine; Phe,phenylalanine; Trp, tryptophan.

to synthesize tryptophan, phenylalanine, andtyrosine. Autotrophs lack a diet and must syn-thesize these and all 17 other amino acids.In plants the shikimate pathway located inthe plastid synthesizes the precursors for thearomatic amino acids. There are two ver-sions of the shikimate pathway: a prokaryotic-style version, which is what occurs in theplant plastid, and a cytosolic-based versionwith different enzymes, as occurs in fungi(45). In plants the plastid has clearly retainedits ancestral ability to synthesize tryptophan,phenylalanine, and tyrosine and supplies theseamino acids to the cytosol (host) (Figure 6).Whether or not the original host possessed ashikimate pathway prior to primary endosym-biosis remains unclear. If it did, all traces arenow lost and the plastid bears sole responsi-bility for this task in members of the red algae,green algae, and plants.

Fe-S Clusters

Fe-S clusters are important prosthetic groupsof various metalloproteins that participate inredox reactions, sensing of iron and oxygen,and catalysis (69). The Fe atom in Fe-S clus-ters is able to take up an electron reversibly,thus providing the required electron carriercapacity. Fe-S–containing proteins are perva-sive in life and ancient; well-known examplesinclude the ferredoxins, NADH dehydroge-nase, and Coenzyme Q. The clusters containdifferent numbers of iron and sulfur depend-ing on cluster type and are coordinated intothe protein through cysteinyl ligands. Syn-thesis and insertion or removal of Fe-S clus-ters into and out of proteins is managed bya number of enzymes, not all of which havebeen identified. At least three different sys-tems for Fe-S cluster formation and inser-tion [iron sulfur cluster (ISC), nitrogen fix-ation (NIF), and mobilization of sulfur (Suf )]are known thus far. Plastids harbor a Suf-type Fe-S cluster formation system homolo-gous to that of cyanobacteria (11, 119). Sufsystem–generated Fe-S clusters are probablyincorporated into a range of plastid proteins


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including ferredoxin and the Rieske iron sul-fur protein.

The original host of the primary plastid en-dosymbiont almost certainly contained an Fe-S cluster formation system, but it was prob-ably not cytosolic. Eukaryotes all appear toform Fe-S clusters, but the initial steps are mi-tochondrial and typically utilize the ISC sys-tem (64). An export machinery translocates

the Fe-S cluster into the cytosol where ma-chinery exists to insert the cluster into apopro-teins. This mitochondrial-based synthesis waslikely obtained with the α-proteobacterialendosymbiont that gave rise to the mito-chondrion (70). The host cell for primaryendosymbiosis likely had this system, and in-deed, plants utilize an ISC system in their mi-tochondria and a Suf system in the plastid (11).

SUMMARY POINTS

1. All plastids ultimately arose from a single prokaryotic endosymbiosis, where acyanobacterium was engulfed and retained by a eukaryotic cell.

2. Eukaryotic endosymbiosis has occurred multiple times, and this process has lead tothe spread of plastids throughout a great diversity of eukaryotes.

3. Further endosymbiotic events continue to occur in nature, and a wide continuum existsbetween temporary symbiotic relationships, stable interdependent partnerships, andthose that are intimately integrated at a molecular-genetic level.

4. Pivotal to the integration of plastids was the establishment of protein delivery systemsthat enabled a shift of genetic control from the organelle to the host nucleus. Prokary-otic endosymbiosis required the generation of a novel protein import system, whereaseukaryotic endosymbionts have co-opted and adapted existing protein translocationsystems to achieve this task for complex plastids.

5. Eukaryotes have gained numerous metabolic capabilities through endosymbiosis, in-cluding but not restricted to photosynthesis. Some of these capabilities were uniqueprokaryotic inventions, and have thus extended the capabilities of eukaryotes. Othersrepresented duplications of existing host pathways, and in many cases rationalizationof redundancy has generated novel chimeric pathways in eukaryotes.

FUTURE ISSUESIn recent years the broadening of genome sequencing programs has encompassed agreater diversity of plastid-bearing eukaryotes, and we are now seeing great advances inour understanding of plastids. This diversity includes the molecular integration of plastidgenomes with those of the host, the mechanisms and trafficking routes of transferred geneproducts on their return journey to the plastid, and the metabolic integration and tradebetween plastids and their diverse hosts. Several key research directions now presentthemselves and are conceivably within greater reach than ever before. (1) Although thephylogenetic affinities between the major eukaryotic lineages are begining to slowlyresolve into focus, considerable controversy continues to surround the question of howmany endosymbiotic events have generated the plastid diversity observed in eukaryotes—notably with respect to the plastids of the Chromalveolates. Resolution of these issues


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is critical to our interpretation of evolution of the diversity we see among plastids inthis group, and our understanding of the frequency and mechanisms of plastid loss.(2) Although our understanding of protein targeting to primary plastids has reached arelatively advanced state, equivalent insight into the mechanisms for targeting proteinsto complex plastids lags behind. These details may be vital in tackling some of the moreinsidious complex plastid-bearing eukaryotes such as the apicomplexan parasites, whereplastid-targeted pathways offer tantalizing possibilities as drug targets for diseases suchas malaria. (3) Genomic analyses have presented some insights into the broader suite ofmetabolic functions of plastids beyond photosynthesis; however, little is known aboutdelivery of the products of these pathways to the host cell, or vice versa. Although thispresents one of the larger challenges to plastid researchers, the realization of this goal isnecessary to provide a full appreciation of the significance of plastid gain in eukaryotesthrough endosymbiosis.

DISCLOSURE STATEMENT

The authors are not aware of any biases that might be perceived as affecting the objectivity ofthis review.

ACKNOWLEDGMENTS

G.M. is an ARC Federation Fellow and Howard Hughes International Scholar and supportedby a Program Grant from the NHMRC. S.B.G. is supported by the ARC grant DP0664097.

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Annual Review ofPlant Biology

Volume 59, 2008Contents

Our Work with Cyanogenic PlantsEric E. Conn � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �1

New Insights into Nitric Oxide Signaling in PlantsAngelique Besson-Bard, Alain Pugin, and David Wendehenne � � � � � � � � � � � � � � � � � � � � � � � � � 21

Plant Immunity to Insect HerbivoresGregg A. Howe and Georg Jander � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 41

Patterning and Polarity in Seed Plant ShootsJohn L. Bowman and Sandra K. Floyd � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 67

Chlorophyll Fluorescence: A Probe of Photosynthesis In VivoNeil R. Baker � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 89

Seed Storage Oil MobilizationIan A. Graham � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �115

The Role of Glutathione in Photosynthetic Organisms:Emerging Functions for Glutaredoxins and GlutathionylationNicolas Rouhier, Stephane D. Lemaire, and Jean-Pierre Jacquot � � � � � � � � � � � � � � � � � � � � �143

Algal Sensory PhotoreceptorsPeter Hegemann � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �167

Plant Proteases: From Phenotypes to Molecular MechanismsRenier A.L. van der Hoorn � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �191

Gibberellin Metabolism and its RegulationShinjiro Yamaguchi � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �225

Molecular Basis of Plant ArchitectureYonghong Wang and Jiayang Li � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �253

Decoding of Light Signals by Plant Phytochromesand Their Interacting ProteinsGabyong Bae and Giltsu Choi � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �281

Flooding Stress: Acclimations and Genetic DiversityJ. Bailey-Serres and L.A.C.J. Voesenek � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �313

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Roots, Nitrogen Transformations, and Ecosystem ServicesLouise E. Jackson, Martin Burger, and Timothy R. Cavagnaro � � � � � � � � � � � � � � � � � � � � � � �341

A Genetic Regulatory Network in the Development of Trichomesand Root HairsTetsuya Ishida, Tetsuya Kurata, Kiyotaka Okada, and Takuji Wada � � � � � � � � � � � � � � � � � �365

Molecular Aspects of Seed DormancyRuth Finkelstein, Wendy Reeves, Tohru Ariizumi, and Camille Steber � � � � � � � � � � � � � � �387

Trehalose Metabolism and SignalingMatthew J. Paul, Lucia F. Primavesi, Deveraj Jhurreea, and Yuhua Zhang � � � � � � � �417

Auxin: The Looping Star in Plant DevelopmentRene Benjamins and Ben Scheres � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �443

Regulation of Cullin RING LigasesSara K. Hotton and Judy Callis � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �467

Plastid EvolutionSven B. Gould, Ross F. Waller, and Geoffrey I. McFadden � � � � � � � � � � � � � � � � � � � � � � � � � � � � �491

Coordinating Nodule Morphogenesis with Rhizobial Infectionin LegumesGiles E.D. Oldroyd and J. Allan Downie � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �519

Structural and Signaling Networks for the Polar Cell GrowthMachinery in Pollen TubesAlice Y. Cheung and Hen-ming Wu � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �547

Regulation and Identity of Florigen: FLOWERING LOCUS T MovesCenter StageFranziska Turck, Fabio Fornara, and George Coupland � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �573

Plant Aquaporins: Membrane Channels with Multiple IntegratedFunctionsChristophe Maurel, Lionel Verdoucq, Doan-Trung Luu, and Veronique Santoni � � � �595

Metabolic Flux Analysis in Plants: From Intelligent Designto Rational EngineeringIgor G.L. Libourel and Yair Shachar-Hill � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �625

Mechanisms of Salinity ToleranceRana Munns and Mark Tester � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �651

Sealing Plant Surfaces: Cuticular Wax Formation by Epidermal CellsLacey Samuels, Ljerka Kunst, and Reinhard Jetter � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �683

Ionomics and the Study of the Plant IonomeDavid E. Salt, Ivan Baxter, and Brett Lahner � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �709

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Alkaloid Biosynthesis: Metabolism and TraffickingJorg Ziegler and Peter J. Facchini � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �735

Genetically Engineered Plants and Foods: A Scientist’s Analysisof the Issues (Part I)Peggy G. Lemaux � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �771

Indexes

Cumulative Index of Contributing Authors, Volumes 49–59 � � � � � � � � � � � � � � � � � � � � � � � �813

Cumulative Index of Chapter Titles, Volumes 49–59 � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �818

Errata

An online log of corrections to Annual Review of Plant Biology articles may be foundat http://plant.annualreviews.org/

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Documents

Plastid Evolution - Sektion Biologie